Our research program seeks to identify molecules and pathways that are important in the normal development and physiology of the ear by discovering and studying gene mutations in mice that disrupt these processes, and to develop mouse models of human hearing disorders and provide them to the scientific community. In Aims 1-3 of this renewal application, we propose to perform in-depth analyses of molecular mechanisms that underlie inner ear pathologies associated with three new mouse mutations chosen because of their unique natures and potentially high impact on hearing research. For Aim 1, we will investigate the hyperspin (hspn) mutation, a spontaneous intragenic deletion of the Slc25a13 gene, which causes a gross malformation of the inner ear. In contrast to the severe auditory phenotype of Slc25a13hspn mutant mice, mice with targeted knockout mutations of this gene have normal ear development and hearing. We will determine whether loss of specific non-transcribed regulatory elements within the hspn deletion are responsible for the Slc25a13hspn mutant phenotype, thereby demonstrating an important role of enhancers in directing early inner ear development and providing a potential explanation for the variable sensorineural hearing loss associated with the human SHFM1 syndrome. For Aim 2, we will exploit the hypomorphic nature of the ENU-induced Tbx1wdml missense mutation to elucidate previously unknown regulatory roles of TBX1 during late stage embryonic development of the inner ear. Tbx1wdml mutant mice are viable with fully formed but dysfunctional inner ears, in contrast to the inner ears of mice with KO alleles of Tbx1, which do not develop beyond the early otocyst stage. The Tbx1wdml mutation causes a reduction in inner ear endolymph volume and provides an entry point for identifying TBX1-regulated genes and transcriptional co-factors that are involved in the development of vestibular dark cells and marginal cells of the stria vascularis, which is much less understood than organ of Corti development. For Aim 3, we will determine the genetic and molecular basis of strain background effects that influence the severity of hearing impairment in mice with the spontaneous vtx mutation of the Atp6v1b1 gene, which is associated with an increased volume of endolymph in the inner ear. Our discovery that genetic background differences explain why the mouse Atp6v1b1vtx mutation (on an MRL strain background) causes deafness whereas a previously reported mouse KO mutation (on a mixed B6/129 strain background) has normal auditory function, may also explain the variability in hearing loss associated with human distal renal tubular acidosis caused by mutations in the ATP6V1B1 gene. For Aim 4, we will use high-throughput exome sequencing and other methods to identify the causative DNA alterations underlying three new mutations we mapped to chromosome regions devoid of known hearing-related genes. We will analyze the hearing loss and characterize the inner ear pathologies associated with these three mutations and with the mutations in three genes we previously identified but have not fully characterized (Mctp1dwnd, Cachd1tow, and Ap1g1fgt).